1. Field of the Invention
The present invention relates to an ion exchange chromatography process for purifying a peptide from a mixture containing said peptide and related impurities, and to an industrial method including such ion exchange chromatography process.
2. Background
For the purification and analysis of proteins and peptides, chromatography is a well-known and widely used method. A number of different chromatographic principles are applied, among these ion exchange chromatography (IEC). The IEC principle includes two different approaches: anion exchange and cation exchange according to the charge of the ligands on the ion exchange resin. A conventional IEC purification process usually consists of one or more: equilibration sections, application or loading sections, wash sections, elution sections, and regeneration sections (cf. Remington""s Pharmaceutical Sciences, Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, or Remington: The Science and Practice of Pharmacy, 19th Edition (1995)).
The main principle of elution in IEC in industrial purification processes is salt component gradients in an aqueous buffer solution at constant pH, either as step or linear gradients (cf. S. Bjxc3x8rn and L. Thim, Activation of Coagulation Factor VII to VIIa, Res. Discl. No. 269, 564-565, 1986). Isocratic elution is possible, but seldom used. Organic solvents or modifiers have occasionally been added to the solutions to keep the protein or peptide on the desired form or just in solution (cf. K. H. Jxc3x8rgensen, Process for Purifying Insulin, U.S. Pat. No. 3,907,676, Sep. 23, 1975; and J. Brange, O. Hallund and E. Sxc3x8rensen, Chemical Stability of Insulin 5. Isolation, Characterisation and Identification of Insulin Transformation Products, Acta Pharm. Nord. 4(4), 223-232, 1992). Also, the change in pH may occasionally be employed to elute the target protein (cf. J. Lamy, J. Lamy, J. Weill, Arch. Biochem. Biophys. 193, 140-149, 1979).
In contrast to the above described IEC techniques for purification of any protein or peptide, consisting of one or more equilibration steps, application or loading steps, wash steps, elution steps, and regeneration steps, the present invention relates to a novel elution technique which is the combination of elution in a solution comprising an organic modifier with the subsequent elution in an aqueous solution at the same or a different pH optionally followed by a regeneration step. The equilibration solution and the sample for application may or may not contain the organic modifier. The elution of the peptide occurs at non-denaturing conditions (in a solution free of organic modifier). Moreover, the elution of the peptide is performed in a single peak.
Accordingly, in a broad aspect the present invention relates to a cation exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the steps of:
a) eluting said related impurities of said mixture in a solution comprising an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a positive local or overall net charge and said related impurities have a local or overall positive net charge which is lower than the positive net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or higher pH-values optionally maintained with a buffer.
In another broad aspect the present invention relates to a cation exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the steps of:
a) eluting said related impurities of said mixture in a solution consisting essentially of an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a positive local or overall net charge and said related impurities have a local or overall positive net charge which is lower than the positive net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or higher pH-values optionally maintained with a buffer.
In another broad aspect the present invention relates to an anion exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the steps of:
a) eluting said related impurities of said mixture in a solution comprising an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a negative local or overall net charge and said related impurities have a local or overall negative net charge which is lower than the negative net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or lower pH-values optionally maintained with a buffer.
In another broad aspect the present invention relates to an anion exchange chromatography process for purifying a peptide from a mixture comprising said peptide and related impurities, comprising the steps of:
a) eluting said related impurities of said mixture in a solution consisting essentially of an organic modifier, water, optionally a salt component and optionally a buffer, at a linear or step gradient or isocratically in salt component, and at pH-values optionally maintained with a buffer so that said peptide has a negative local or overall net charge and said related impurities have a local or overall negative net charge which is lower than the negative net charge of said peptide so as to remove said related impurities,
b) subsequently, eluting said peptide by a step or linear change to an aqueous solvent optionally with a salt component, at the same or lower pH-values optionally maintained with a buffer.
In the above aspects of the present process the elution in step a) could also be considered a washing step of related impurities.
The elution of the peptide in step b) occurs at non-denaturing conditions (in a solution free of organic modifier). Thus, the peptide is eluted to an aqueous solution with a solution comprising water and optionally a salt component, an acid or base, and/or a buffer, but without the presence of an organic modifier.
In one embodiment of the present invention the ratio of organic modifier to water, on a weight percent basis, is from 1:99 to 99:1, such as from 1:99 to 80:20, 20:80 to 80:20, 30:70 to 70:30, 35:50 to 50:35, or 40:50 to 50:40. Each of these ranges constitutes an alternative embodiment of the present invention.
In further embodiments of the present invention the organic modifier is selected from C1-6-alkanol, C1-6-alkenol or C1-6-alkynol, urea, guanidine, or C1-6-alkanoic acid, such as acetic acid, C2-6-glycol, C3-7-polyalcohol including sugars, preferably C1-6-alkanol and C2-6-glycol, more preferably methanol, ethanol, propanols and butanols and hexyl glycols, most preferably ethanol and 2-propanol. Each of these organic modifiers constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the salt component in step a) is selected from any organic or inorganic salt and mixtures thereof, preferably NaCl, KCl, NH4Cl, CaCl2, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, ammonium citrate, sodium sulphate, potassium sulphate, ammonium sulphate, calcium acetate or mixtures thereof, most preferred sodium acetate, potassium acetate, ammonium acetate, NaCl, NH4Cl, KCl. Each of these salt components constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the gradient in salt component in step a) is a step gradient in the salt component.
In a further embodiment of the present invention the salt component in step a) is present in a step concentration selected from the range of 0.1 mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, more preferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg. Each of these ranges constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the salt component gradient in step a) is a linear gradient in salt component.
In a further embodiment of the present invention the salt component in step a) is present in a linear concentration selected from 0.1 mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, more preferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg. Each of these linear concentrations constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention no salt component is present in step a.
In a further embodiment of the present invention the salt component in step b) is selected from any organic or inorganic salt, preferably NaCl, KCl, NH4Cl, CaCl2, sodium acetate, potassium acetate, ammonium acetate, sodium citrate, potassium citrate, ammonium citrate, sodium sulphate, potassium sulphate, ammonium sulphate, calcium acetate or mixtures thereof, most preferred sodium acetate, potassium acetate, ammonium acetate, NaCl, NH4Cl, KCl. Each of these salt components constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the salt component in step b) is present in a concentration selected from the range of 0.1 mmol/kg to 3000 mmol/kg, preferably 1 mmol/kg to 1000 mmol/kg, more preferably 5 mmol/kg to 500, most preferably 20 mmol/kg to 300 mmol/kg. Each of these ranges constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention no salt component is present in step b).
In a further embodiment of the present invention the buffer in step a) or b) is independently selected from citrate buffers, phosphate buffers, tris buffers, borate buffers, lactate buffers, glycyl glycin buffers, arginine buffers, carbonate buffers, acetate buffers, glutamate buffers, ammonium buffers, glycin buffers, alkylamine buffers, aminoethyl alcohol buffers, ethylenediamine buffers, tri-ethanol amine, imidazole buffers, pyridine buffers and barbiturate buffers and mixtures thereof, preferably citric acid, sodium citrate, sodium phosphate, phosphoric acid, glutamic acid, sodium glutamate, glycin, sodium carbonate, potassium citrate, potassium phosphate, potassium glutamate, potassium carbonate, tris-hydroxymethyl amino methane and boric acid and mixtures thereof. Each of these buffers constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the buffer in step a) is present in a concentration selected from the range of 0.1 mmol/kg to 500 mmol/kg, preferably 1 mmol/kg to 200 mmol/kg, more preferably 5 mmol/kg to 100 mmol/kg, most preferably 10 mmol/kg to 50 mmol/kg. Each of these ranges constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention the buffer in step b) is present in a concentration selected from the range of 0.1 mmol/kg to 1000 mmol/kg, preferably 1 mmol/kg to 400 mmol/kg, most preferably 50 mmol/kg to 200 mmol/kg. Each of these ranges constitutes an alternative embodiment of the present invention.
In a further embodiment of the present invention no buffer is present in step a).
In a further embodiment of the present invention no buffer is present in step b).
In a further embodiment of the present invention the peptide to be purified is selected from polypeptides, oligopeptides, proteins, receptors, vira, as well as homologues, analogues and derivatives thereof, preferably glucagon, hGH, insulin, aprotinin, FactorVII, TPA, FactorVIIa, FFR-FactorVIIa, heparinase, ACTH, Heparin Binding Protein, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon-like peptide-1, glucagon-like peptide-2, insulin-like growth factor-1, insulin-like growth factor-2, fibroblast growth factors, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opiods, DPP IV, interleukins, immunoglobulins, complement inhibitors, serpin protease inhibitors, cytokines, cytokine receptors, PDGF, tumor necrosis factors, tumor necrosis factors receptors, growth factors and analogues as well as derivatives thereof, more preferably glucagon, hGH, insulin, aprotinin, FactorVII, FactorVIIa, FFR-FactorVIIa, heparinase, glucagon-like peptide-1, glucagon-like peptide-2 and analogues as well as derivatives thereof, such as Val8GLP-1(7-37), Thr8GLP-1 (7-37), Met8GLP-1 (7-37), Gly8GLP-1(7-37), Val8GLP-1(7-36) amide, Thr8GLP-1(7-36) amide, Met8GLP-1(7-36) amide, Gly8GLP-1(7-36) amide, Arg34GLP-1(7-37), human insulin, and B28IsoAsp insulin. Each of these peptides constitutes an alternative embodiment of the present invention.